Understanding of the phylogeny and interrelationships of the genera within the order 'Enterobacteriales' has proven difficult using the 16S rRNA gene and other single-gene or limited multi-gene approaches. In this work, we have completed comprehensive comparative genomic analyses of the members of the order 'Enterobacteriales' which includes phylogenetic reconstructions based on 1548 core proteins, 53 ribosomal proteins and four multilocus sequence analysis proteins, as well as examining the overall genome similarity amongst the members of this order. The results of these analyses all support the existence of seven distinct monophyletic groups of genera within the order 'Enterobacteriales'. In parallel, our analyses of protein sequences from the 'Enterobacteriales' genomes have identified numerous molecular characteristics in the forms of conserved signature insertions/deletions, which are specifically shared by the members of the identified clades and independently support their monophyly and distinctness. Many of these groupings, either in part or in whole, have been recognized in previous evolutionary studies, but have not been consistently resolved as monophyletic entities in 16S rRNA gene trees. The work presented here represents the first comprehensive, genome-scale taxonomic analysis of the entirety of the order 'Enterobacteriales'. On the basis of phylogenetic analyses and the numerous identified conserved molecular characteristics, which clearly distinguish members of the order 'Enterobacteriales' and the seven reported clades within this order, a proposal is made here for the order Enterobacterales ord. nov. which consists of seven families: Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov.

Phylogenomic analyses and molecular signatures for the class Halobacteria and its two major clades: a proposal for division of the class Halobacteria into an emended order Halobacteriales and two new orders, Haloferacales ord. nov. and Natrialbales ord. nov., containing the novel families Haloferacaceae fam. nov. and Natrialbaceae fam. nov. The Halobacteria constitute one of the largest groups within the Archaea. The hierarchical relationship among members of this large class, which comprises a single order and a single family, has proven difficult to determine based upon 16S rRNA gene trees and morphological and physiological characteristics. This work reports detailed phylogenetic and comparative genomic studies on .100 halobacterial (haloarchaeal) genomes containing representatives from 30 genera to investigate their evolutionary relationships. In phylogenetic trees reconstructed on the basis of 32 conserved proteins, using both neighbour-joining and maximum-likelihood methods, two major clades (clades A and B) encompassing nearly two-thirds of the sequenced haloarchaeal species were strongly supported. Clades grouping the same species/genera were also supported by the 16S rRNA gene trees and trees for several individual highly conserved proteins (RpoC, EF-Tu, UvrD, GyrA, EF-2/EF-G). In parallel, our comparative analyses of protein sequences from haloarchaeal genomes have identified numerous discrete molecular markers in the form of conserved signature indels (CSI) in protein sequences and conserved signature proteins (CSPs) that are found uniquely in specific groups of haloarchaea. Thirteen CSIs in proteins involved in diverse functions and 68 CSPs that are uniquely present in all or most genome-sequenced haloarchaea provide novel molecular means for distinguishing members of the class Halobacteria from all other prokaryotes. The members of clade A are distinguished from all other haloarchaea by the unique shared presence of two CSIs in the ribose operon protein and small GTP-binding protein and eight CSPs that are found specifically in members of this clade. Likewise, four CSIs in different proteins and five other CSPs are present uniquely in members of clade B and distinguish them from all other haloarchaea. Based upon their specific clustering in phylogenetic trees for different gene/protein sequences and the unique shared presence of large numbers of molecular signatures, members of clades A and B are indicated to be distinct from all other haloarchaea because of their uniquely shared evolutionary histories. Based upon these results, it is proposed that clades A and B be recognized as two new orders, Natrialbales ord. nov. and Haloferacales ord. nov., within the class Halobacteria, containing the novel families Natrialbaceae fam. nov. and Haloferacaceae fam. nov. Other members of the class Halobacteria that are not members of these two orders will remain part of the emended order Halobacteriales in an emended family Halobacteriaceae. INTRODUCTIONThe class Halobacteria, members of which will be referred t...

Non-aureus staphylococci (NAS), the microorganisms most frequently isolated from bovine milk worldwide, are a heterogeneous group of numerous species. To establish their importance as a group, the distribution of individual species needs to be determined. In the present study, NAS intramammary infection (IMI) was defined as a milk sample containing ≥1,000 cfu/mL in pure or mixed culture that was obtained from a cohort of cows assembled by the Canadian Bovine Mastitis Research Network. Overall, 6,213 (6.3%) of 98,233 quarter-milk samples from 5,149 cows and 20,305 udder quarters were associated with an NAS IMI. Of the 6,213 phenotypically identified NAS isolates, 5,509 (89%) were stored by the Canadian Bovine Mastitis Research Network Mastitis Pathogen Collection and characterized using partial sequencing of the rpoB housekeeping gene, confirming 5,434 isolates as NAS. Prevalence of each NAS species IMI was estimated using Bayesian models, with presence of a specific NAS species as the outcome. Overall quarter-level NAS IMI prevalence was 26%. The most prevalent species causing IMI were Staphylococcus chromogenes (13%), Staphylococcus simulans (4%), Staphylococcus haemolyticus (3%), Staphylococcus xylosus (2%), and Staphylococcus epidermidis (1%). The prevalence of NAS IMI as a group was highest in first-parity heifers and was evenly distributed throughout cows in parities ≥2. The IMI prevalence of some species such as S. chromogenes, S. simulans, and S. epidermidis differed among parities. Overall prevalence of NAS IMI was 35% at calving, decreased over the next 10 d, and then gradually increased until the end of lactation. The prevalence of S. chromogenes, Staphylococcus gallinarum, Staphylococcus cohnii, and Staphylococcus capitis was highest at calving, whereas the prevalence of S. chromogenes, S. haemolyticus, S. xylosus, and S. cohnii increased during lactation. Although the overall prevalence of NAS IMI was similar across barn types, the prevalence of S. simulans, S. xylosus, S. cohnii, Staphylococcus saprophyticus, S. capitis, and Staphylococcus arlettae IMI was higher in tiestall barns; the prevalence of S. epidermidis IMI was lowest; and the prevalence of S. chromogenes and Staphylococcus sciuri IMI was highest in bedded-pack barns. Staphylococcus simulans, S. epidermidis, S. xylosus, and S. cohnii IMI were more prevalent in herds with intermediate to high bulk milk somatic cell count (BMSCC) and S. haemolyticus IMI was more prevalent in herds with high BMSCC, whereas other common NAS species IMI were equally prevalent in all 3 BMSCC categories. Distribution of NAS species IMI differed among the 4 regions of Canada. In conclusion, distribution differed considerably among NAS species IMI; therefore, accurate identification (species level) is essential for studying NAS epidemiology.

Non-aureus staphylococci (NAS) are the most frequently isolated pathogens from intramammary infection (IMI) in dairy cattle. Virulence factors (VFs) and mechanisms by which NAS cause IMI are not fully known. Herein, we analyzed the distribution of 191 VFs in 441 genomes of 25 NAS species, after classifying VFs into functional categories: adherence (n = 28), exoenzymes (n = 21), immune evasion (n = 20), iron metabolism (n = 29), and toxins (n = 93). In addition to establishing VF gene profiles, associations of VF genes between and among functional categories were computed, revealing distinctive patterns of association among VFs for various NAS species. Associations were also computed for low, medium, and high somatic cell count (SCC) and clinical mastitis (CM) isolates, demonstrating distinctive patterns of associations for low SCC and CM isolates, but no differences between high SCC and CM isolates. To determine whether VF distributions had any association with SCC or CM, various clustering approaches, including complete linkages, Ward clustering, and t-distributed stochastic neighbor embedding, were applied. However, no clustering of isolates representing low SCC, medium SCC, or high SCC or CM was identified. Regression analysis to test for associations with individual VF functional categories demonstrated that each additional toxin and host immune evasion gene increased the odds of having high SCC or CM, although an overall increase in the number of VFs was not associated with increased SCC or occurrence of CM. In conclusion, we established comprehensive VF gene profiling, determined VF gene distributions and associations, calculated pathogenic potentials of all NAS species, and detected no clear link between VF genes and mastitis. IMPORTANCE Non-aureus staphylococci (NAS) are the most frequently isolated pathogens from milk in dairy cattle worldwide. The virulence factors (VFs) and mechanisms by which these bacteria cause udder infection are not fully known. We determined the distribution and associations of 191 VFs in 25 NAS species and investigated the relationship between VFs and disease. Although the overall number of VFs was not associated with disease severity, increasing numbers of toxin and host immune evasion genes specifically were associated with more severe disease outcomes. These findings suggest that the development of disease and the interactions of VFs with the host are complex and determined by the interplay of genes rather than just the presence of virulence genes. Together, our results provide foundational genetic knowledge to other researchers to design and conduct further experiments, focusing on understanding the synergy between VFs and roles of individual NAS species in IMI and characterizing species-specific effects on udder health.

Despite considerable efforts to control bovine mastitis and explain its causes, it remains the most costly and common disease of dairy cattle worldwide. The role and impact of non-aureus staphylococci (NAS) in udder health are not entirely understood. These Gram-positive bacteria have become the most frequently isolated group of bacteria in milk samples of dairy cows and are associated with (mild) clinical and subclinical mastitis. Different species and strains of NAS differ in their epidemiology, pathogenicity, virulence, ecology and host adaptation, and antimicrobial resistance profiles. They have distinct relationships with the microbiome composition of the udder and may also have protective effects against other mastitis pathogens. Some appear to persist on the skin and in the teat canal and udder, while others seem to be transient residents of the udder from the environment. Analyzing genotypic and phenotypic differences in individual species may also hold clues to why some appear more successful than others in colonizing the udder. Understanding species-level interactions within the microbiome and its interactions with host genetics will clarify the role of NAS in bovine mastitis and udder health.